Cardiac Sarcomere Protein Quality Control in Health and Disease

健康和疾病中的心脏肌节蛋白质量控制

基本信息

批准号：
10621229
负责人：
JONATHAN A KIRK
金额：
$ 53.92万
依托单位：
LOYOLA UNIVERSITY CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-04-01 至 2027-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10621229
关键词：
Actins Autophagosome BAG3 gene Binding Biological Assay Biophysics Cardiac Cardiac Myocytes Cells Client Complex Data Depressed mood Disease Functional disorder Future Generations Grant Half-Life Health Heart Heart failure Heat-Shock Proteins 70 Heat-Shock Response Human Impairment In Vitro Knockout Mice Knowledge Lasers Lysosomes Mechanical Stress Mediating Mental Depression Methods Modeling Modification Molecular Chaperones Mus Muscle Cells Myocardial Infarction Myosin ATPase Nature Neonatal Pathway interactions Patients Phenotype Process Proteins Publishing Quality Control Rattus Regulation Relaxation Role Sarcomeres Sedimentation process Signal Transduction Stimulus Stress Structure System Therapeutic Transgenic Mice Tropomyosin Ubiquitin Ubiquitination Ventricular Visualization Work cell motility cost gene therapy improved in vivo induced pluripotent stem cell live cell imaging misfolded protein new therapeutic target protein degradation protein protein interaction proteotoxicity recruit response spatiotemporal ubiquitin isopeptidase ubiquitin ligase ultra high resolution

项目摘要

Cardiomyocytes are essentially non-renewing, so proteotoxicity from dysregulated Protein Quality Control (PQC) is intimately associated with heart failure. The proteins that comprise the cardiac sarcomere are responsible for force generation in the myocyte, and this constant mechanical stress uniquely predisposes them to misfolding. Despite PQC’s central role in heart failure, and the particular vulnerability of the sarcomere to misfolding, the PQC mechanisms that maintain the cardiac sarcomere are almost entirely unknown. This is a critical knowledge gap that we will address in this proposal. Our past work described a z-disc localized complex, anchored by the co- chaperone Bcl2-associated athanogene-3 (BAG3), that was essential for sarcomere PQC. Z-disc BAG3 levels were depressed in human heart failure (HF) and correlated with decreased sarcomeric force-generating capacity (Fmax). Similarly, cardio-myocyte specific inducible BAG3 KO mice had increased ubiquitination of sarcomeric proteins that remained integrated in the lattice, reducing force generation. Importantly, BAG3 gene therapy in a mouse HF model reversed this phenotype, indicating the potential of targeting sarcomere PQC to improve contractile function. In this renewal we will address the central hypothesis that sarcomere PQC occurs via sarcomere-localized pathways and depression of these systems in HF due to BAG3 instability results in accumulation of ubiquitinated proteins that induce dysfunction. In Aim 1 we will Explore the spatiotemporal organization of the key steps in sarcomere PQC. We will use super-resolution live cell imaging in neonatal rat ventricular myocytes (NRVMs) and human iPSC-CMs to visualize the spatiotemporal interplay between BAG3, autophagosomes, lysosomes, the z-disc, and BAG3-clients at baseline and in response to various stress and stimuli, such as heat shock, localized laser damage, hypertrophic signaling, and depressed BAG3 levels. In Aim 2 we will identify the functional consequences of sarcomeric protein ubiquitination. We will use in vivo and in vitro approaches to modulate sarcomere protein ubiquitination and assess the impact on sarcomere function with biophysical assays (force- Ca2+ relationship, tension cost, in vitro motility assay, super-relaxed state, co-sedimentation). In Aim 3 we will discover the regulation of BAG3 in the cardiomyocyte and how it is altered in heart failure. We will use several transgenic mouse lines and a myocardial infarction induced heart failure model, to discover the interplay between HSP70, BAG3, heart failure, and sarcomere PQC. We expect to identify new methods to stabilize BAG3 in the failing heart as a possible therapeutic strategy. These 3 aims establish a foundational understanding of sarcomere PQC, functional consequences of its misregulation, and how it can be modulated in vivo.

心肌细胞本质上是非更新的，因此蛋白质失调的蛋白毒性质量控制（PQC）与心力衰竭密切相关。完成的蛋白质心脏肌膜负责肌细胞中的力产生，并且这种常数机械压力独特地使他们容易折叠。尽管PQC在心力衰竭中起着核心作用，肌膜错误折叠的特殊脆弱性，维持的PQC机制心脏肌膜几乎完全未知。这是我们将要的关键知识差距在此提案中的地址。我们过去的工作描述了一个Z二烟的局部化合物，由共同锚定伴侣BCl2相关的achanogene-3（BAG3），这对于Saromere PQC至关重要。 Z式 BAG3水平在人体心力衰竭（HF）中降低，并与肉类降低相关力产能（FMAX）。同样，心肌细胞特异性诱导Bag3 KO小鼠有增加肉瘤蛋白的泛素化增加，该蛋白保持在晶格中，减少力量一代。重要的是，小鼠HF模型中的BAG3基因疗法逆转了这种表型，表明靶向肌节PQC以提高收缩功能的潜力。在这个续约中我们将解决以下中心假设，即肌节PQC通过肌膜定位的途径发生由于BAG3不稳定性导致HF中这些系统的抑郁导致积累诱导功能障碍的泛素化蛋白。在AIM 1中，我们将探索时空 Saromere PQC的关键步骤的组织。我们将在新生大鼠心室肌细胞（NRVM）和人IPSC-CM可视化时空 Bag3，自噬体，溶酶体，Z-DISC和BAG3客户在基线和BAG3之间的相互作用响应各种压力和刺激，例如热冲击，局部激光损伤，肥厚信号传导和凹陷的Bag3水平。在AIM 2中，我们将确定功能后果肉瘤蛋白泛素化。我们将使用体内和体外方法调节肌膜蛋白质泛素化和评估生物物理测定对肌节功能的影响（力 - CA2+关系，张力成本，体外运动评估，超级省力状态，共同报道）。目标 3我们将发现心肌细胞中BAG3的调节及其在心力衰竭中的改变。我们将使用几种转基因小鼠系和心肌梗死诱导的心力衰竭模型，发现HSP70，BAG3，心力衰竭和肌节PQC之间的相互作用。我们希望确定新方法将BAG3稳定在失败的心脏中，作为可能的治疗策略。这3 目的建立对肌节PQC的基本理解，其功能后果正直，以及如何在体内调节它。